The role of hormone-evoked mitochondrial calcium increases in the pathogenesis of

描述(申请人提供)：酒精滥用对身体几乎每一个组织都有有害影响，是全球发病率和死亡率的主要原因。酒精对细胞功能的急性作用是完全可逆的，而长期酒精中毒可导致不可逆的组织损伤。不可逆细胞损伤的发生和发展的分子机制仍不清楚。在这一应用中，我们提出了一种假设，即肌醇磷脂依赖的信号通路的适应性变化在酒精诱导的组织损伤的发病机制中起着关键作用。我们的初步数据表明，以含乙醇的液体饮食(即DeCarli-Lieber)喂养大鼠60天，可以增强肝脏对磷脂酶C-β偶联激素的敏感性。与配对的对照组相比，低浓度激素的加入引起酒精喂养动物分离的肝细胞胞浆钙离子更持续的增加。此外，来自酒精动物的细胞在低激素刺激下有更大的三磷酸肌醇(InsP3)形成增加，这表明长期饮酒改变了磷脂酶C-？活性。正常情况下，细胞内钙的增加激活了线粒体的生理功能，使ATP的形成与利用相匹配；然而，长时间或不适当的钙增加也会导致基质钙超载和线粒体功能障碍。线粒体损伤是慢性酒精中毒患者和酒精滥用动物模型的共同特征。线粒体功能障碍可能会增加组织对其他类型的损伤或凋亡刺激的易感性。这可能在慢性酒精中毒患者中尤其重要，因为慢性酒精中毒患者的TNFa水平升高，这是一种促炎细胞因子，通过线粒体依赖的途径引起肝细胞凋亡。在这项建议中，我们将(1)研究酒精诱导的肌醇磷脂依赖的信号通路的变化，(2)确定InsP3依赖的钙增加对对照组和慢性酒精喂养大鼠肝细胞线粒体钙水平和能量代谢的影响，(3)确定钙激活激素对酒精喂养大鼠及其配对喂养对照组大鼠肝细胞TNFa诱导的细胞凋亡的影响。拟议的研究将为乙醇持续存在引起的适应性反应以及相关的肝脏损害后果提供新的见解。

DESCRIPTION (provided by applicant): Alcohol abuse has deleterious affects on almost every tissue in the body and is a major cause of morbidity and mortality worldwide. The acute actions of alcohol on cellular function are fully reversible whereas long-term alcohol intoxication can lead to irreversible tissue damage. The molecular mechanisms contributing to the onset and progression of irreversible cell injury are still poorly understood. In this application, we propose to investigate the hypothesis that adaptive changes in the phosphoinositide-dependent signaling pathway play a key role in the pathogenesis of alcohol-induced tissue injury. Our preliminary data indicate that feeding rats an ethanol-containing liquid diet (i.e., DeCarli-Lieber) for 60 days enhances the liver' sensitivity to hormones coupled to phospholipase C-¿. The addition of low concentrations of hormones evoked more sustained cytosolic calcium increases in hepatocytes isolated from alcohol-fed animals compared to their pairfed controls. Moreover, cells from alcoholic animals had a larger increase in inositol-1,4,5-trisphosphate (InsP3) formation following low hormone stimulation suggesting that chronic alcohol consumption altered phospholipase C-¿ activity. Normally, cytosolic calcium increases activate mitochondrial physiology to match ATP formation with utilization; however prolonged or inappropriate calcium increases can also lead to matrix calcium overload and mitochondrial dysfunction. Mitochondrial damage is a common feature observed in chronic alcoholic patients and animal models of alcohol abuse. Mitochondrial dysfunction may increase the tissues' susceptibility to other types of injury or apoptotic stimuli. This may be particularly important in chronic alcoholics that have elevated levels of TNFa, a proinflammatory cytokine, which evokes apoptotic cell death in hepatocytes through the mitochondrial-dependent pathway. In this proposal, we will (1) characterize the alcohol-induced alterations in phosphoinositide-dependent signaling pathway, (2) determine the effects of InsP3-dependent calcium increases on mitochondrial calcium levels and energy metabolism in control and chronically ethanol-fed rats and (3) determine the effects Ca2+-moblizing hormones on TNFa-induced apoptosis in hepatocytes from alcohol-fed rats and their pair-fed controls. The proposed studies will provide new insights into the adaptive responses evoked by the sustained presence of ethanol and the associated injurious consequences for the liver.